The Auto-inhibitory Function of Importin α Is Essentialin Vivo

Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/ karyopherin alpha and beta. The importin a subunit recognizes classical NLS sequences, and the importin beta subunit directs the complex to the nuclear pore. Recent work shows that the N-terminal importin beta binding (IBB) domain of importin alpha regulates N-LS-cargo binding in the absence of importin beta in vitro. To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin alpha protein. These mutants dissect the two functions of the N-terminal IBB domain, importin beta binding and autoinhibition. One of these importin alpha mutations, A3, decreases auto-inhibitory function without impacting binding to importin beta or the importin a export receptor, Cse1p. We used this mutant to show that the auto-inhibitory function is essential in vivo and to provide evidence that this auto-inhibitory-defective importin a remains bound to NLS-cargo within the nucleus. We propose a model where the auto-inhibitory activity of importin alpha is required for N-LS-cargo release and the subsequent Cse1p-dependent recycling of importin a to the cytoplasm.